Efficient screening method of influenza virus candidate vaccine strain and application thereof

Abstract

The invention relates to an efficient screening method and application of an influenza virus candidate vaccine strain, and the efficient screening method comprises the following steps: (1) diluting a mother generation influenza virus to be screened by using a virus maintenance solution, adding the diluted mother generation influenza virus into a culture plate containing canine kidney cells, adsorbing the canine kidney cells by the influenza virus, and discarding a virus solution in the culture plate; (2) covering the surfaces of the cells with a maintenance medium in a liquid state, cooling and solidifying the maintenance medium, and inversely placing the maintenance medium in an incubator to culture viruses; and (3) selecting virus spots, mixing the selected virus spots with a buffer solution, inoculating the obtained mixture into chick embryos and / or canine kidney cells, and culturing to obtain the candidate vaccine strain of the influenza virus. The method provided by the invention is simple and easy to operate, short in time consumption, low in cost, few in passage times and not easy to mutate, and can achieve the purpose of quickly and effectively screening out influenza virus vaccine candidate strains with excellent growth characteristics from mother generation virus seeds in a short time.

Description

Technical Field

[0001] The present invention relates to the field of virology, and in particular to an efficient screening method for candidate influenza virus vaccine strains and an application thereof. Background Art

[0002] Influenza virus is an important respiratory pathogen. Infecting humans can cause high fever, fatigue, headache, cough, serious respiratory diseases and even death. It belongs to the genus Influenza virus of the family Orthomyxoviridae. Its genome is a single-stranded negative-strand segmented RNA fragment. According to the differences in antigenicity of nucleoprotein (NP) and matrix protein (M) in the virus functional proteins, influenza viruses can be divided into four types: A, B, C and D. Every year, influenza A and B viruses co-circulate worldwide, causing seasonal pandemics that can infect millions of people, of which about 290,000 to 650,000 people die.

[0003] Currently, the most effective way to prevent and treat influenza virus infection is still vaccination, but the high incidence of seasonal influenza infection and the antigenic drift of the virus lead to the mismatch between the vaccine strain and the epidemic strain, which makes the vaccine ineffective in the current season, which has always been a major problem in influenza prevention and treatment. Because influenza virus subtypes are numerous and mutate rapidly, countries around the world have not been able to establish effective herd immunity, resulting in the return of influenza viruses every year, which continues to threaten global public health security.

[0004] The surface of influenza virus particles is wrapped by a lipid capsule from the host cell membrane, and the membrane surface is inlaid with two glycoproteins, hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins are the main antigens on the surface of influenza viruses and are also the main effective ingredients of current influenza vaccines. There are currently many technical solutions for influenza vaccines, including inactivated vaccines, attenuated live vaccines, recombinant protein vaccines, and mRNA vaccines, among which the earliest and most widely used are inactivated vaccines. In recent years, with the development of biotechnology, influenza vaccines using new vaccination methods such as nasal spray immunization and microneedle vaccination have also appeared. However, the mainstream influenza vaccine products currently available on the market mainly use an inactivated lysis solution, that is, a technical route of lysing and purifying hemagglutinin after culturing influenza viruses on chicken embryos or cell matrices. The advantages of low cost, stability, safety, reliability, and fast production time of inactivated vaccines are still irreplaceable, so the yield of effective antigens of influenza viruses, namely hemagglutinin, during the cultivation process is an important factor affecting vaccine yield and quality.

[0005] The production of influenza virus hemagglutinin is mainly affected by both internal and external factors. The sequence of the viral genome determines the strength of the role played by the viral functional protein in the replication process, which in turn determines the growth characteristics of the virus. The stronger the growth characteristics of the virus and the faster the replication speed, the higher the hemagglutinin production in the same culture time. In addition, external factors such as the environmental temperature, pH, dissolved oxygen ratio, etc. of the virus culture will also affect the growth and replication of the virus. Under the premise of a fixed virus culture environment, the growth characteristics of the virus strain itself determine the production of influenza virus hemagglutinin per unit time.

[0006] CN116218791A discloses a rabies virus screening method based on in vivo and in vitro crossover, which increases the virus titer by multiple passages after in vivo and in vitro screening. CN105671002A discloses a method for constructing and screening a high-yield vaccine strain of avian influenza virus cells, which obtains a high-yield vaccine strain through reverse genetics and site-directed mutagenesis operations; in addition, there is a limiting dilution method for continuous passage on cells or chicken embryos, etc.

[0007] The above methods can improve the virus titer and virus yield to varying degrees, but there are still many problems in industrial application: 1) Some methods have many subcultures and long subculture times, and cannot guarantee the single sequence of the virus strain after subculture. Mutations may occur, leading to changes in antigenicity; 2) Reverse genetics technology has a certain operation threshold and a low success rate. Generally, reverse genetics technology involves processes such as vector construction, homologous recombination, point mutation, and virus rescue. It has many operation steps, a long cycle, and a low success rate, and is not suitable for technology promotion; 3) In vivo and in vitro cross-screening is costly and time-consuming, and animal experiments are required. Due to the role of the immune system in animals, viruses are usually subjected to stress screening after infection and are prone to mutations to adapt to the individual environment. Mutations may occur, leading to changes in antigenicity.

[0008] In summary, how to quickly screen influenza virus candidate vaccine strains with high titer, high hemagglutinin production and no mutation, and apply them to the construction of influenza virus candidate vaccine strains library has become one of the urgent problems to be solved in this field. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides an efficient screening method for influenza virus candidate vaccine strains and its application, which can quickly and effectively screen candidate vaccine strains with excellent growth characteristics and antigen production from the parent virus working seed batch under the premise of very few passages.

[0010] To achieve this object, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a method for efficiently screening candidate influenza virus vaccine strains, the method comprising the following steps:

[0012] (1) diluting the parent influenza virus to be screened using a virus maintenance solution, adding the diluted virus to a culture plate containing canine kidney cells, allowing the influenza virus to adsorb to the canine kidney cells, and discarding the virus solution in the culture plate;

[0013] (2) Cover the cell surface with liquid maintenance medium, cool and solidify the maintenance medium, and invert it in an incubator to culture the virus;

[0014] (3) picking virus spots, mixing them with a buffer, and inoculating them into chicken embryos and / or canine kidney cells for culture to obtain the influenza virus candidate vaccine strain.

[0015] In order to obtain influenza virus strains with high antigenicity and no mutation, as influenza virus candidate vaccine strains, it is necessary to reduce the number of passages and improve screening efficiency. The present invention provides a screening method that can quickly and effectively screen candidate vaccine strains with excellent growth characteristics and antigen yield from parent virus working seed batches under the premise of very few passages, and can be applicable to the screening of most influenza viruses. The chicken embryo used in the present invention is an 8-10 day old SPF chicken embryo, but the present invention is not limited to the influenza virus vaccine candidate strains used for chicken embryo vaccine production, and can also be used for candidate strains of influenza virus cultured based on cell matrix.

[0016] Preferably, the influenza virus comprises any one of influenza A virus, influenza B virus, influenza C virus or influenza D virus.

[0017] Preferably, the virus maintenance solution in step (1) is a DMEM culture medium containing 0.2% to 1% (for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%) w / v bovine serum albumin and 1 to 5 μg / mL (for example, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL or 5 μg / mL, etc.) pancreatic enzyme.

[0018] Preferably, the pancreatic enzyme comprises TPCK-treated pancreatic enzyme and / or recombinant pancreatic enzyme.

[0019] Preferably, the step (1) of diluting with a virus maintenance solution comprises: using the virus maintenance solution to perform a 10-fold volume gradient dilution, from 10 3 Dilute to 10 8 times, specifically 10 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 .

[0020] Preferably, the method for preparing the culture plate containing canine kidney cells in step (1) comprises: digesting and counting the canine kidney cells in the logarithmic growth phase and in good condition, and culturing the culture plate at a density of 4×10 5 ~6×10 5 / hole (for example, 4×10 5 , 4.5×10 5 4.8×10 5 , 5×10 5 , 5.2×10 5 , 5.5×10 5 or 6×10 5 The cells were inoculated in a 6-well cell culture plate and incubated at 35 to 40°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C) containing 4% to 6% (e.g., 4%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, or 6%) CO 2 The cells are cultured in an incubator for 18 to 24 hours (for example, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, etc.) until a dense monolayer of cells is formed, and the culture medium is discarded.

[0021] Preferably, in step (1), the temperature of the influenza virus adsorption to canine kidney cells is 34-37° C. (for example, 34° C., 35° C., 35.5° C., 36° C. or 37° C., etc.), and the time is 1-2 h (for example, 1 h, 1.2 h, 1.4 h, 1.5 h, 1.6 h, 1.8 h or 2 h, etc.).

[0022] Preferably, after discarding the virus solution in the culture plate in step (1), the step further includes washing the cells 2 to 3 times with PBS buffer.

[0023] Preferably, the maintenance medium in step (2) is a MEM medium containing 0.5% to 1% (for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%) agarose, 0.2% to 1% (for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%) w / v bovine serum albumin and 1 to 5 μg / mL (for example, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL or 5 μg / mL, etc.) pancreatic enzyme.

[0024] The bovine serum albumin in the maintenance medium of the present invention can maintain the activity of cells for a certain period of time, and the MEM medium can use a commonly used product in the art. After screening and comparing multiple culture media, 2×MEM serum-free medium is used as a maintenance medium component, which can significantly promote the growth of influenza virus.

[0025] Preferably, the preparation method of the maintenance culture medium in step (2) comprises: preparing liquid A and liquid B respectively, heating and melting liquid A before use, and when the temperature of liquid A drops to 25-35°C (for example, it can be 25°C, 27°C, 29°C, 30°C, 31°C, 33°C or 35°C, etc.), mixing liquid A and liquid B in a volume ratio of 1: (0.8-1.2), and using them immediately after preparation, wherein the liquid A contains 1% to 2% (for example, it can be 1%, 1.2%, 1.4%, 1.5%, The B solution is a PBS buffer containing 0.4% to 2% (for example, 0.4%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%) agarose, and the B solution is a MEM culture medium containing 0.4% to 2% (for example, 0.4%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%) w / v bovine serum albumin and 2 to 10 μg / mL (for example, 2 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL or 10 μg / mL, etc.) pancreatin.

[0026] The specific point values ​​of the above (0.8-1.2) can be selected from 0.8, 0.9, 1, 1.1 or 1.2, etc.

[0027] The maintenance medium with specific components provided by the present invention can be applied to the cultivation of most influenza viruses, and the maintenance medium and virus maintenance liquid provided by the present invention do not contain antibiotics. The present invention realizes the full mixing of agarose and other components by preparing the components of the maintenance medium into liquid A and liquid B respectively, and preparing and using them immediately when using them, thereby improving the uniformity of the maintenance medium and being more conducive to the growth of influenza viruses.

[0028] Preferably, the agarose comprises low melting point agarose.

[0029] Preferably, the pancreatic enzyme comprises TPCK-treated pancreatic enzyme and / or recombinant pancreatic enzyme.

[0030] Preferably, the conditions for culturing the virus in step (2) are: 33-37°C (for example, 33°C, 34°C, 35°C, 36°C or 37°C, etc.) containing 4%-6% (for example, 4%, 4.5%, 4.8%, 5%, 5.2%, 5.5% or 6%) CO 2 The cells are cultured in an incubator for 48 to 120 hours (for example, 48 hours, 60 hours, 80 hours, 84 hours, 90 hours, 100 hours, or 120 hours).

[0031] In the present invention, the virus culture time is 48 to 120 hours. If the culture time is more than 48 hours, multiple virus monoclones can show differences in growth characteristics and the replication amount of virus particles reaches a certain amount. The time to end the culture is determined according to the growth of the virus monoclonal clones, and the best time is when the diameters of different monoclonal strains are obviously different, but the edges of adjacent monoclonal clones are not in contact. If the culture time is less than 120 hours, it can be ensured that the sequences of the obtained monoclonal strains are consistent and mutations are not likely to occur.

[0032] Preferably, when the virus cultured in step (2) is influenza A virus, the culture temperature is 35-37°C (for example, 35°C, 35.5°C, 36°C, 36.5°C or 37°C, etc.).

[0033] Preferably, when the virus cultured in step (2) is influenza B virus, the culture temperature is 33-35°C (for example, 33°C, 33.5°C, 34°C, 34.5°C or 35°C, etc.).

[0034] Preferably, the picking of virus spots in step (3) specifically includes: picking virus spots with a diameter of 1 to 4 mm (for example, 1 mm, 2 mm, 2.5 mm, 3 mm or 4 mm, etc.) and clear edges, and vertically separating the solid maintenance culture medium and the bottom cells.

[0035] The present invention obtains influenza viruses with stronger growth and replication capabilities by picking virus spots with larger diameters, and at the same time, all cultures in the longitudinal space are taken out, thereby retaining the viruses to the greatest extent.

[0036] Preferably, the buffer in step (3) comprises PBS buffer.

[0037] Preferably, step (3) further includes the step of precooling the buffer solution before picking the virus spots and mixing them with the buffer solution.

[0038] The present invention ensures that the selected monoclonal virus particles have good activity by precooling the buffer solution, thereby improving the selection success rate. Normal temperature will cause the virus particles to be quickly inactivated.

[0039] Preferably, the culture conditions in step (3) are: 33-37°C (for example, 33°C, 34°C, 35°C, 36°C or 37°C, etc.) containing 4%-6% (for example, 4%, 4.5%, 4.8%, 5%, 5.2%, 5.5% or 6%) CO 2 The cells are cultured in an incubator for 48 to 96 hours (for example, 48 hours, 50 hours, 60 hours, 70 hours, 72 hours, 75 hours, 80 hours, 90 hours, or 96 hours, etc.).

[0040] Preferably, when the culture in step (3) is for culturing influenza A virus, the culture temperature is 35-37°C (for example, 35°C, 35.5°C, 36°C, 36.5°C or 37°C, etc.).

[0041] Preferably, when the culture in step (3) is for culturing influenza B virus, the culture temperature is 33-35°C (for example, 33°C, 33.5°C, 34°C, 34.5°C or 35°C, etc.).

[0042] Preferably, after the culturing in step (3), the HA and NA sequences of the virus are sequenced and confirmed, and strains without amino acid mutations are selected as candidate influenza virus vaccine strains for library construction.

[0043] In a second aspect, the present invention provides the use of the efficient screening method for influenza virus candidate vaccine strains as described in the first aspect in screening highly antigenic influenza viruses.

[0044] The influenza virus candidate vaccine strain obtained by screening using the method provided by the present invention has high antigenicity, high hemagglutinin production, and high influenza virus antigenicity.

[0045] Preferably, the highly antigenic influenza virus exhibits high hemagglutinin production.

[0046] In a third aspect, the present invention provides use of the efficient screening method for influenza virus candidate vaccine strains as described in the first aspect in the preparation of influenza virus vaccines.

[0047] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The method provided by the present invention is simple and easy to operate, short in time, low in cost, requires few passages, is not prone to mutation, and applies the plaque test for determining virus titer to the field of isolating monoclonal vaccine candidate strains and increasing the antigen effective component (i.e., hemagglutinin production), thereby achieving the purpose of quickly and effectively screening influenza virus vaccine candidate strains with excellent growth characteristics from parental virus strains in a short period of time.

[0050] (2) Compared with the reverse genetics method commonly used in the field of influenza virus research to modify viruses, the method provided by the present invention is time-saving and low-cost, and does not require steps such as constructing expression vectors and rescuing recombinant viruses. It only takes 5 to 7 days to complete a round of screening and obtain preliminary screening results.

[0051] (3) Compared with screening influenza viruses in chicken embryos, the viruses screened by the method provided by the present invention have the characteristics of single sequence and not easy to mutate. Since chicken embryos have natural screening pressure, the virus has a certain selection adaptability in chicken embryos, and its hemagglutinin sequence is very easy to mutate.

[0052] (4) Compared with the commonly used limiting dilution method for virus screening, the method provided by the present invention can intuitively observe the differences in virus growth characteristics, and the virus strains screened are all monoclonal strains. A single spot is a collection of single virus particles that infect surrounding healthy cells and replicate, which is not easy to mutate and has a single sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a graph showing the results of the hemagglutinin content determination in a 250 mL shake flask test of H1N1 strains before and after screening using the method of Example 1.

[0054] Figure 2 This is a graph showing the results of the hemagglutinin content determination in a 250 mL shake flask test after the H1N1 strains were screened using the methods of Example 1 and Comparative Example 1.

[0055] Figure 3 This is a graph showing the results of measuring the hemagglutinin content in the harvest fluid cultured in a 10 L scale bioreactor before and after the H1N1 strain was screened using the method of Example 1.

[0056] Figure 4 This is a plaque diagram of H1N1 strains formed on canine kidney cells before and after screening using the method of Example 1. DETAILED DESCRIPTION

[0057] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0059] Reagents used in the following examples:

[0060] Bovine serum albumin (BSA): purchased from MP Biomedicals;

[0061] TPCK-treated trypsin: purchased from Sigma;

[0062] Recombinant pancreatic enzyme: purchased from Beijing Kangshi;

[0063] DMEM medium: purchased from Gibco;

[0064] Low melting point agarose: purchased from Invitrogen;

[0065] 2xMEM culture medium: purchased from Gibco, catalog number 11935046.

[0066] Example 1

[0067] This embodiment provides an efficient screening method for influenza virus candidate vaccine strains, comprising the following steps:

[0068] (1) Digest and count canine kidney cells in the logarithmic growth phase and in good condition at a density of 5×10 5 Cells were inoculated into 6-well cell culture plates and incubated at 38°C with 5% CO 2 The cells were cultured in an incubator for 20 h until a dense monolayer of cells was formed, and the culture medium was discarded.

[0069] (2) The virus maintenance solution was used to perform a 10-fold volume gradient dilution of the parent influenza A virus to be screened, from 10 3 Dilute to 10 8 The virus was diluted to 0.6 times and then added to a culture plate containing canine kidney cells. The influenza virus was adsorbed to the canine kidney cells at 35°C for 1.5 hours. The virus solution in the culture plate was discarded and the cells were washed 2 to 3 times with PBS buffer. The virus maintenance solution was DMEM culture medium containing 0.6% w / v bovine serum albumin and 3 μg / mL TPCK-treated trypsin.

[0070] (3) Cover the cell surface with liquid maintenance medium, cool and solidify the maintenance medium, and invert it in an incubator to culture the virus. The virus culture conditions are: 36°C with 5% CO 2 Culture in an incubator for 80 h;

[0071] Among them, the preparation method of the maintenance culture medium includes: preparing liquid A and liquid B respectively, heating and melting liquid A before use, and when the temperature of liquid A drops to 30°C, mixing liquid A and liquid B in a volume ratio of 1:1, and preparing and using them immediately, wherein the liquid A is a PBS buffer containing a mass fraction of 1.5% low-melting point agarose, and the liquid B is a MEM culture medium containing 1.2% w / v bovine serum albumin and 6 μg / mL TPCK-treated trypsin.

[0072] (4) Virus spots with a diameter of 1 to 4 mm and clear edges were picked out, the solid maintenance medium and the bottom cells were vertically separated and mixed with pre-cooled PBS buffer, and inoculated into SPF-grade chicken embryos at 36°C with 5% CO 2 Culture in an incubator for 70 h.

[0073] (5) Sequencing to confirm the HA and NA sequences of the virus, and selecting strains without amino acid mutations as candidate influenza A virus vaccine strains for library construction.

[0074] Example 2

[0075] This embodiment provides an efficient screening method for influenza virus candidate vaccine strains, comprising the following steps:

[0076] (1) Digest and count canine kidney cells in the logarithmic growth phase and in good condition at a density of 4×10 5 Cells were inoculated into 6-well cell culture plates and incubated at 40°C with 4% CO 2 The cells were cultured in an incubator for 18 h until a dense monolayer of cells was formed, and the culture medium was discarded.

[0077] (2) The virus maintenance solution was used to perform a 10-fold volume gradient dilution of the parent influenza A virus to be screened, from 10 3 Dilute to 10 8 The virus was diluted to 500 μg / mL and then added to a culture plate containing canine kidney cells. The influenza virus was adsorbed to the canine kidney cells at 37°C for 1 hour. The virus solution in the culture plate was discarded and the cells were washed 2 to 3 times with PBS buffer. The virus maintenance solution was DMEM culture medium containing 0.2% w / v bovine serum albumin and 5 μg / mL recombinant trypsin.

[0078] (3) Cover the cell surface with liquid maintenance medium, cool and solidify the maintenance medium, and invert it in an incubator to culture the virus. The virus culture conditions are: 37°C with 6% CO 2 Culture in an incubator for 48 h;

[0079] Among them, the preparation method of the maintenance culture medium includes: preparing liquid A and liquid B respectively, heating and melting liquid A before use, and when the temperature of liquid A drops to 35°C, mixing liquid A and liquid B in a volume ratio of 1:0.8, and preparing and using them immediately, wherein the liquid A is a PBS buffer containing a mass fraction of 1% low-melting point agarose, and the liquid B is a MEM culture medium containing 2% w / v bovine serum albumin and 2 μg / mL recombinant trypsin.

[0080] (4) Virus spots with a diameter of 1 to 4 mm and clear edges were picked out, the solid maintenance medium and the bottom cells were vertically separated and mixed with pre-cooled PBS buffer, and inoculated into canine kidney cells at 37°C with 6% CO 2 Culture in an incubator for 48 h.

[0081] (5) Sequencing to confirm the HA and NA sequences of the virus, and selecting strains without amino acid mutations as candidate influenza A virus vaccine strains for library construction.

[0082] Example 3

[0083] This embodiment provides an efficient screening method for influenza virus candidate vaccine strains, comprising the following steps:

[0084] (1) Digest and count canine kidney cells in logarithmic growth phase and in good condition at a density of 6×10 5 Cells were inoculated into 6-well cell culture plates and incubated at 35°C with 6% CO 2 The cells were cultured in an incubator for 24 h until a dense monolayer of cells was formed, and the culture medium was discarded.

[0085] (2) The virus maintenance solution was used to perform a 10-fold volume gradient dilution of the parent influenza A virus to be screened, from 10 3 Dilute to 10 8 The virus was diluted to 100 μg / mL and then added to a culture plate containing canine kidney cells. The influenza virus was adsorbed to the canine kidney cells at 34°C for 2 h, the virus solution in the culture plate was discarded, and the cells were washed 2 to 3 times with PBS buffer; wherein the virus maintenance solution was DMEM culture medium containing 1% w / v bovine serum albumin and 1 μg / mL TPCK-treated trypsin.

[0086] (3) Cover the cell surface with liquid maintenance medium, cool and solidify the maintenance medium, and invert it in an incubator to culture the virus. The conditions for culturing the virus are: 35°C with 4% CO 2 Culture in an incubator for 120 h;

[0087] Among them, the preparation method of the maintenance culture medium includes: preparing liquid A and liquid B respectively, heating and melting liquid A before use, and when the temperature of liquid A drops to 25°C, mixing liquid A and liquid B in a volume ratio of 1:1.2, and preparing and using them immediately, wherein the liquid A is a PBS buffer containing a mass fraction of 2% low-melting point agarose, and the liquid B is a MEM culture medium containing 0.4% w / v bovine serum albumin and 10 μg / mL recombinant trypsin.

[0088] (4) Virus spots with a diameter of 1 to 4 mm and clear edges were picked out, the solid maintenance medium and the bottom cells were vertically separated and mixed with pre-cooled PBS buffer, and inoculated into canine kidney cells at 35°C with 4% CO 2 Culture in an incubator for 120 h.

[0089] (5) Sequencing to confirm the HA and NA sequences of the virus, and selecting strains without amino acid mutations as candidate influenza A virus vaccine strains for library construction.

[0090] Example 4

[0091] This embodiment provides an efficient screening method for candidate influenza virus vaccine strains. The only difference from Embodiment 1 is that in step (3), the maintenance culture medium does not contain bovine serum albumin, and the proportion of bovine serum albumin is allocated by other components in proportion.

[0092] Example 5

[0093] This embodiment provides an efficient screening method for candidate influenza virus vaccine strains, which is different from Example 1 only in that the preparation method of the maintenance culture medium in step (3) is: low melting point agarose, bovine serum albumin, TPCK-treated pancreatic enzyme and MEM culture medium are directly mixed to obtain a MEM culture medium containing 0.75% low melting point agarose, 0.6% w / v bovine serum albumin and 3 μg / mL TPCK-treated pancreatic enzyme by mass fraction.

[0094] Example 6

[0095] This embodiment provides an efficient screening method for candidate influenza virus vaccine strains, which differs from Embodiment 1 only in that the virus is cultured for 40 hours in step (3).

[0096] Example 7

[0097] This embodiment provides an efficient screening method for candidate influenza virus vaccine strains, which differs from Embodiment 1 only in that the virus is cultured for 130 hours in step (3).

[0098] Comparative Example 1

[0099] The present comparative example provides a method for screening candidate influenza virus vaccine strains, wherein the parent influenza A virus is subjected to chicken embryo adaptation and passage for several times and optionally cell adaptation and passage for several times to obtain candidate influenza virus vaccine strains, and the steps of cell passage and chicken embryo passage are performed with reference to the Standard Operating Procedure of the National Influenza Center.

[0100] Test Example 1

[0101] In this test example, the method provided in Examples 1-7 was used to screen a working seed batch of a candidate H1N1 vaccine strain A / Victoria / 4897 / 2022 (IVR-238, from the World Health Organization) for the 2023-2024 northern hemisphere epidemic season to obtain a candidate influenza virus vaccine strain. The Reed-Muench method was used to detect the virus titer (half tissue infection dose, TCID 50 / mL), and the HA and NA sequences of the candidate strains and the IVR-238 genome before screening were sequenced (Sanger method) and compared by Shanghai Sangon Biotechnology Co., Ltd. The results of virus titration and sequencing are shown in Table 1.

[0102] In addition, in this test example, the selected virus strains and IVR-238 were inoculated at a density of 3.5×10 6 / mL suspended canine kidney cells, small-scale suspension culture in 250mL cell shake flasks, final concentration of recombinant trypsin was 3μg / mL, culture environment was 34℃, 5% CO 2 , 120rpm suspension incubator, samples were taken at 48h and 72h after inoculation to detect the hemagglutinin content, and the results are shown in Table 1. The comparison results of the hemagglutinin content of SX1204, SX1205, SX1206, SX1207, SX1208, SX1211, SX1212 (after screening) and IVR-238 (before screening) are shown in Figure 1 Among them, Examples 2-7 only list one candidate strain virus species, and the candidate strain virus species can be used as a representative of the virus after screening of the corresponding experimental group, which is the average level of the corresponding experimental group.

[0103] Table 1

[0104]

[0105]

[0106] As can be seen from Table 1, the virus titer of the candidate strains of the virus vaccine after screening can be increased from 7.5lgTCID 50 / mL up to 9.1lgTCID 50 / mL. Under the same culture conditions and virus inoculation amount, the hemagglutinin content of the virus harvest fluid of the vaccine candidate strain in a 250mL suspension cell shake flask after screening and culture for 48 hours can be increased from 11.7μg / mL to a maximum of 28.2μg / mL, and the hemagglutinin content of the virus harvest fluid after culture for 72 hours can be increased from 15.4μg / mL to a maximum of 38.4μg / mL, and the hemagglutinin content is significantly improved.

[0107] Comparing Example 1 with Example 4, it can be seen that the bovine serum albumin in the maintenance medium plays an important role in the growth of the virus; comparing Example 1 with Example 5, it can be seen that the maintenance medium prepared by the present invention using the ready-to-use method can improve the uniformity of the medium and help the growth of the virus; comparing Example 1 with Example 6 and Example 7, it can be seen that if the virus culture time is too short or too long, the yield of the virus hemagglutinin obtained by screening will be reduced.

[0108] In addition, in this test example, SX1204, SX1211, the virus species SX1 (adapted to chicken embryos and passed down twice, adapted to cells and passed down once), SX2 (adapted to chicken embryos and passed down twice, adapted to cells and passed down once), SX3 (adapted to chicken embryos and passed down once, adapted to cells and passed down four times), SX4 (adapted to chicken embryos and passed down once, adapted to cells and passed down five times), SX7 (adapted to chicken embryos and passed down twice) screened by the method provided in Comparative Example 1 were sampled and tested for hemagglutinin content at 72h, 96h and 120h after inoculation, respectively, with reference to the aforementioned method. The results are shown in Table 2 and Figure 2 As shown, Figure 2 The hemagglutinin content of each time group from left to right is SX1204, SX1211, SX1, SX2, SX3, SX4 and SX7. The hemagglutinin content of the influenza virus vaccine candidate strain obtained by the screening method provided by the present invention is higher than that of the influenza virus vaccine candidate strain obtained by other methods.

[0109] Table 2

[0110]

[0111]

[0112] Test Example 2

[0113] In this test example, the screened virus strain SX1204 was inoculated into a 10L bioreactor at an inoculation volume of MOI 0.001, and the unscreened IVR-238 was inoculated into a 10L bioreactor at an inoculation volume of MOI 0.001 and 0.01, respectively. The cell density was 3.5×10 6 / mL, the final concentration of recombinant trypsin was 3μg / mL, the culture temperature was controlled at 34°C, the stirring speed was 60rpm, the pH was 7.0, and the dissolved oxygen ratio was 50%. Samples were taken at 72h, 96h and 120h after inoculation, and the virus harvest liquid was centrifuged at 1000rpm for 5min to detect the hemagglutinin content in the supernatant. The results are shown in Table 3 and Figure 3 As shown, Figure 3 The time groups from left to right are SX1204 (0.001MOI), IVR-238 (0.001MOI), and IVR-238 (0.01MOI).

[0114] Table 3

[0115]

[0116] As can be seen from Table 3, in a 10L bioreactor, under the same culture conditions, the hemagglutinin content of the virus harvest fluid of the screened strain can be increased from 22.5μg / mL to 39.0μg / mL at most, and the hemagglutinin yield is significantly improved.

[0117] Test Example 3

[0118] In this test example, the screened virus strain SX1204 and the unscreened IVR-238 were cultured on adherent canine kidney cells under the same conditions and time, and the immunostaining method was used for color development. Figure 4 As shown, the left picture is IVR-238, and the right picture is SX1204. The diameter of the plaque formed by SX1204 is larger than that of IVR-238, indicating that SX1204 has a stronger growth and replication ability.

[0119] In summary, the screening method provided by the present invention is simple and easy to operate, short in time, low in cost, requires few passages, is not prone to mutation, and applies the plaque test for determining virus titer to the field of isolating monoclonal vaccine candidate strains and increasing the antigen effective component (i.e., hemagglutinin production), which can achieve the purpose of quickly and effectively screening out influenza virus vaccine candidate strains with excellent growth characteristics from the parent virus strains in a short time.

[0120] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for efficiently screening candidate influenza virus vaccine strains, characterized in that: The efficient screening method comprises the following steps: (1) diluting the parent influenza virus to be screened using a virus maintenance solution, adding the diluted virus to a culture plate containing canine kidney cells, allowing the influenza virus to adsorb to the canine kidney cells, and discarding the virus solution in the culture plate; (2) Cover the cell surface with liquid maintenance medium, cool and solidify the maintenance medium, and invert it in an incubator to culture the virus; (3) picking virus spots, mixing them with a buffer, and inoculating them into chicken embryos and / or canine kidney cells for culture to obtain the influenza virus candidate vaccine strain.

2. The efficient screening method according to claim 1, characterized in that: The influenza virus includes any one of influenza A virus, influenza B virus, influenza C virus or influenza D virus.

3. The efficient screening method according to claim 1 or 2, characterized in that: The virus maintenance solution in step (1) is a DMEM culture medium containing 0.2% to 1% w / v bovine serum albumin and 1 to 5 μg / mL pancreatin; Preferably, the pancreatic enzyme comprises TPCK-treated pancreatic enzyme and / or recombinant pancreatic enzyme; Preferably, the step (1) of diluting with a virus maintenance solution comprises: using the virus maintenance solution to perform a 10-fold volume gradient dilution, from 10 3 Dilute to 10 8 times; Preferably, the method for preparing the culture plate containing canine kidney cells in step (1) comprises: digesting and counting the canine kidney cells in the logarithmic growth phase and in good condition, and culturing the culture plate at a density of 4×10 5 ~6×10 5 The cells were inoculated into 6-well cell culture plates and cultured in an incubator at 35-40°C with 4%-6% CO2 for 18-24 hours until a dense monolayer of cells was formed, and the culture medium was discarded. Preferably, in step (1), the temperature of the influenza virus adsorption to the canine kidney cells is 34-37° C. and the time is 1-2 h; Preferably, after discarding the virus solution in the culture plate in step (1), the step further includes washing the cells 2 to 3 times with PBS buffer.

4. The efficient screening method according to any one of claims 1 to 3, characterized in that: The maintenance medium in step (2) is MEM medium containing 0.5% to 1% agarose by mass, 0.2% to 1% w / v bovine serum albumin and 1 to 5 μg / mL pancreatin; Preferably, the preparation method of the maintenance culture medium in step (2) comprises: preparing liquid A and liquid B respectively, heating and melting liquid A before use, and when the temperature of liquid A drops to 25-35° C., mixing liquid A and liquid B in a volume ratio of 1:(0.8-1.2), and using them immediately after preparation, wherein the liquid A is a PBS buffer solution containing 1% to 2% agarose by mass fraction, and the liquid B is a MEM culture medium containing 0.4% to 2% w / v bovine serum albumin and 2-10 μg / mL pancreatin; Preferably, the agarose comprises low melting point agarose; Preferably, the pancreatic enzyme comprises TPCK-treated pancreatic enzyme and / or recombinant pancreatic enzyme.

5. The efficient screening method according to any one of claims 1 to 4, characterized in that: The conditions for culturing the virus in step (2) are: culturing in an incubator at 33-37°C containing 4%-6% CO2 for 48-120 hours; Preferably, when the virus cultured in step (2) is influenza A virus, the culture temperature is 35-37°C; Preferably, when the virus cultured in step (2) is influenza B virus, the culture temperature is 33-35°C.

6. The efficient screening method according to any one of claims 1 to 5, characterized in that: The step (3) of picking up virus spots specifically includes: picking up virus spots with a diameter of 1 to 4 mm and clear edges, and vertically separating the solid maintenance medium and the bottom cells; Preferably, the buffer in step (3) comprises PBS buffer; Preferably, step (3) further comprises the step of precooling the buffer solution before the virus spots are picked and mixed with the buffer solution; Preferably, the culture conditions in step (3) are: culture in an incubator at 33-37°C containing 4%-6% CO2 for 48-96 hours; Preferably, when the culture in step (3) is for culturing influenza A virus, the culture temperature is 35-37° C.; Preferably, when the culture in step (3) is for culturing influenza B virus, the culture temperature is 33-35°C.

7. The efficient screening method according to any one of claims 1 to 6, characterized in that: After the culturing in step (3), the HA and NA sequences of the virus are sequenced and confirmed, and strains without amino acid mutations are selected as candidate influenza virus vaccine strains for library construction.

8. Use of the method for efficiently screening candidate influenza virus vaccine strains according to any one of claims 1 to 7 in screening highly antigenic influenza viruses.

9. The use according to claim 8, characterized in that: The highly antigenic influenza virus exhibits high hemagglutinin production.

10. Use of the method for efficiently screening candidate influenza virus vaccine strains according to any one of claims 1 to 7 in preparing influenza virus vaccines.

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